Therapeutic nucleosides

ABSTRACT

The use of a 1,3-oxathiolane nucleoside analogue and pharmaceutically acceptable derivatives thereof for the treatment of hepatitis B virus infections is disclosed. Pharmaceutical formulations are also provided.

The present invention relates to the use of a1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine derivative andphysiologically functional derivatives thereof for the treatment ofhepatitis B viral infections.

Hepatitis B virus (HBV) is a viral pathogen of major worldwideimportance. HBV is most common in Asian Countries, and prevalent insub-Saharan Africa. The virus is etiologically associated with primaryhepatocellular carcinoma and is thought to cause 80% of the world'sliver cancer. In the United States more than ten thousand people arehospitalized for HBV illness each year, an average of 250 die withfulminant disease. The United States currently contains an estimatedpool of 500,000-1 million infectious carriers. Chronic active hepatitiswill develop in over 25% of carriers and often progresses to cirrhosis.It is estimated that 5000 people die from HBV-related cirrhosis eachyear in the U.S.A. and that perhaps 1000 die from HBV-related livercancer. Even when a universal HBV vaccine is in place, the need foreffective anti-HBV compounds will continue. The large reservoir ofpersistently infected carriers, estimated at 220 million worldwide, willreceive no benefit from vaccination and will continue at high risk forHBV-induced liver disease. This carrier population serves as the sourceof infection of susceptible individuals perpetuating the instance ofdisease particularly in endemic areas or high risk groups such as i.v.drug abusers and homosexuals. Thus, there is a great need for effectiveantiviral agents, both to control the chronic infection and reduceprogression to hepatocellular carcinoma.

Clinical effects of infection with HBV range from headache, fever,malaise, nausea, vomiting, anorexia and abdominal pains. Replication ofthe virus is usually controlled by the immune response, with a course ofrecovery lasting weeks or months in humans, but infection may be moresevere leading to persistent chronic liver disease as outlined above. In“Viral Infections of Humans” (second edition, Ed. Evans, A. S. (1982)Plenum Publishing Corporation, New York), Chapter 12 describes theetiology of viral hepatitis infections.

European Patent Specification 0 382 526 discloses certain1,3-oxathio-lane nucleoside analogues which are effective in inhibitingthe replication of human immunodeficiency virus (HIV).

Since the priority date of this patent application, the following itemshave been published: Doong, S. L. et al., Inhibition of the replicationof hepatitis B virus in vitro by 2′,3′-dideoxy-3′-thiacytidine andrelated analogs, Proc. Natl. Acad. Sci. USA, 88 (19), 8495-9 (1991);Liotta, D. C. and Choi, W. B., Synthesis of BCH-189 and relatedcompounds, PCT Appl. WO 91/11186; Soudeyns, H. et al., Anti-humanimmunodeficiency virus type 1 activity and in vitro toxicity of2′-deoxy-3′-thiacytidine (BCH-189), Antimicrob. Agents Chemother., 35(7), 1386-90 (1991) and Choi, W. B. et al., In situ complexation directsthe stereochemistry of N-glycosylation in the synthesis of thialanyl anddioxolanyl nucleoside analogs, J. Am. Chem. Soc., 113 (24), 9377-9(1991).

We have now surprisingly found that a1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine derivative of formulaI

namely 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine or apharmaceutically acceptable salt, ester or other physiologicallyfunctional derivatives thereof have potent activity against HBV.

It should be noted that the compound of formula (I) contains two chiralcenters and therefore exists in the form of two pairs of optical isomers(i.e. enantiomers) and mixtures thereof including racemic mixtures.Thus, the compound of formula (I) may be either a cis or a trans isomeror mixtures thereof. Each cis and trans isomer can exist as one of twoenantiomers or mixtures thereof including racemic mixtures. All suchisomers and mixtures thereof including racemic mixtures and tautomericforms of the compound of formula (I) are within the scope of theinvention. The cis isomers of the compound of formula (I) are preferred.

According to one feature of the present invention we provide thecompound of formula (I) or a physiologically functional derivativethereof for use in the treatment or prophylaxis of a hepatitis B virusinfection. According to a further feature of the present invention weprovide the use of the compound of formula (I) or a physiologicallyfunctional derivative thereof, in the manufacture of a medicament forthe treatment or prophylaxis of a hepatitis B virus infection.

In a further aspect of the present invention there is included a methodfor the treatment or prophylaxis of a hepatitis B virus infection in ahost, for example, a mammal such as a human which comprises treating thehost with a therapeutically effective amount of the compound of formula(I) or a physiologically functional derivative thereof.

By “physiologically functional derivative” is meant a pharmaceuticallyacceptable salt, amide, ester or salt of an ester of the compound offormula (I) or any other compound which upon administration to therecipient, is capable of providing (directly or indirectly) the saidcompound of formula (I) or an active metabolite or residue thereof.

Preferred esters in accordance with the invention include carboxylicacid esters in which the non-carbonyl moiety of the carboxylic acidportion of the ester grouping is selected from straight or branchedchain alkyl e.g. n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g.methoxymethyl), arylalkyl (e.g. benzyl), aryloxyalkyl (e.g.phenoxymethyl), and aryl (e.g. phenyl); sulfonate esters such as alkyl-or arylalkylsulfonyl (e.g. methanesulfonyl); amino acid esters (e.g.L-valyl or L-isoleucyl); dicarboxylic acid esters (e.g. hemisuccinate);and 5′-mono- di- or tri-phosphate esters. The phosphate esters may befurther esterified by, for example, a C₁₋₂₀ alcohol or reactivederivative thereof, or by a 2,3-di(C₆₋₂₄)acyl glycerol. Any alkyl moietypresent in such esters advantageously contains 1 to 18 carbon atoms,particularly 1 to 4 carbon atoms. Any aryl moiety present in such estersadvantageously comprises a phenyl group optionally substituted e.g. byhalogen, C₁₋₄ alkyl, C₁₋₄ alkoxy or nitro.

The above-mentioned pharmaceutically acceptable amides of the compoundof formula (I) include those derivatives wherein the cytosine aminogroup is present in the form of an amide, e.g. —NHCOR where R is C₁₋₆alkyl or aryl (e.g. phenyl optionally substituted by halogen, C₁₋₄alkyl, C₁₋₄ alkoxy, nitro or hydroxyl).

Examples of pharmaceutically acceptable salts according to the inventioninclude base salts, e.g. derived from an appropriate base, such asalkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts,ammonium and NX₄ ⁺ (wherein X is C₁₋₄ alkyl). Pharmaceuticallyacceptable acid addition salts include salts of organic carboxylic acidssuch as acetic, lactic, tartaric, malic, isethionic, lactobionic andsuccinic acids; organic sulfonic acids such as methanesulfonic,ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids andinorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamicacids.

The amount of the compound of formula (I) (hereinafter also referred toas the “active ingredient”) or physiologically functional derivativethereof which is required in a medication to achieve the desired effectwill depend on a number of factors, in particular the specificapplication, the nature of the particular compound used, the mode ofadministration and the condition of the patient. In general a suitabledose will be in the range of 3.0 to 120 mg per kilogram body weight ofthe recipient per day, preferably in the range of 6 to 90 mg perkilogram body weight per day and most preferably in the range 15 to 60mg per kilogram body weight per day. The desired dose is preferablypresented as two, three, four, five, six or more sub-doses administeredat appropriate intervals throughout the day. These sub-doses may beadministered in unit dosage forms, for example, containing 10 to 1500mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of activeingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active ingredient of from about 1 to about75 μM, preferably about 2 to 50 μM, most preferably about 3 to about 30μM. This may be achieved, for example, by the intravenous injection of a0.1 to 5% solution of the active ingredient, optionally in saline, ororally administered as a bolus containing about 1 to about 100 mg/kg ofthe active ingredient. Desirable blood levels may be maintained by acontinuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or byintermittent infusions containing about 0.4 to about 15 mg/kg of theactive ingredient.

In the manufacture of a medicament according to the invention,hereinafter referred to as a “formulation”, the compound of formula (I)or a physiologically functional derivative thereof herein as “activeingredient”, is typically admixed with, inter alia, one or morepharmaceutically acceptable carriers or excipients and optionally othertherapeutic agents.

The formulations include those suitable for oral, rectal, nasal, topical(including transdermal, buccal and sublingual), vaginal or parenteral(including subcutaneous, intramuscular, intravenous and intradermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive ingredient with the carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and thenif necessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surface-active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for examplehydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Formulations suitable for oral use as described above may also includebuffering agents designed to neutralize stomach acidity. Such buffersmay be chosen from a variety of organic or inorganic agents such as weakacids or bases admixed with their conjugated salts.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Compositions suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound I) in an optionally buffered,aqueous solution or 2) dissolved and/or dispersed in an adhesive or 3)dispersed in a polymer. A suitable concentration of the active compoundis about 1% to 35%, preferably about 3% to 15%. As one particularpossibility, the active compound may be delivered from the patch byelectrotransport or iontophoresis as generally described inPharmaceutical Research, 3 (6), 318 (1986).

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injections solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, as liposomes or other microparticulatesystems which are designed to target the compounds to blood componentsor one or more organs. The formulations may be presented in unit-dose ormulti-dose sealed containers, for example, ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents.

The compound of formula I may be prepared for example by:

-   a) reacting an optionally protected 5-F-cytosine compound with a    1,3-oxathiolane of formula (IIA)

wherein R₁ is hydrogen or a hydroxy protecting group and L is a leavinggroup; or

-   b) reacting a compound of formula (IIB)

(wherein R₁ is as defined above and R₁ ^(a) is an amino protectinggroup) with a fluorinating agent serving to introduce a fluorine atom inthe 5-position of the cytosine ring; or

-   c) reacting a compound of formula (IIC)

(wherein R₁ is as defined above) with an agent serving to convert theoxo group in the 4-position of the uracil ring to an amino group; anyremaining protecting groups being removed for example by acid or basehydrolysis to produce the desired product.

With regard to process a), the hydroxy protecting group includesprotecting groups such as acyl (e.g. acetyl), arylacyl (e.g. benzoyl orsubstituted benzoyl), trityl or monomethoxytrityl, benzyl or substitutedbenzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) ordiphenylmethylsilyl. The 5-F-cytosine compound may be optionallyprotected with silyl, e.g. trimethyl silyl groups. Such groups may beremoved in conventional manner. The leaving group L is a leaving grouptypical of those known in the art of nucleoside chemistry e.g. halogensuch as chlorine or bromine, alkoxy such as methoxy or ethoxy or acylsuch as acetyl or benzoyl.

The reaction in process a) may be effected in an organic solvent (e.g.1,2-dichloroethane or acetonitrile) in the presence of a Lewis acid suchas stannic chloride or trimethylsilyl triflate.

Compounds of formula IIA may be obtained from a suitably protected2-hydroxyacetaldehyde of formula (III),

R₁OCH₂CHO  (III)

wherein R₁ is defined above, as described in Can. J. Research, 8, 129(1933) and European Patent Specification 0 382 526. Reaction ofcompounds of formula (III) with a mercaptoacetal HSCH₂CH(OR)₂, wherein Ris C₁₋₄ alkoxy such as HSCH₂CH(OC₂H₅)₂, known in the art (Chem. Ber.85:924-932, 1952), yields compounds of formula IIA wherein L is OR(alkoxy) e.g. methoxy or ethoxy. Alternatively, compounds of formulaIIA, wherein L is alkoxy, may be converted to compounds of formula IIAwherein L is halogen or acyl by methods known in the art of carbohydratechemistry.

Compounds of formula (III) may be prepared from 1,2-0-isopropylideneglycerol by introduction of R₁ (e.g. trisubstituted silyl, benzyl ortrityl) and removal of the isopropylidene group with mild acid (e.g.aqueous formic or acetic acid) or zinc bromide in acetonitrile, followedby oxidation of the alcohol group with aqueous periodate.

With regard to process b), the 5-fluoro substituent may be introduced bymethods known in the art (X. J. Robins, et al., in Nucleic AcidChemistry, Part 2, L. B. Townsend and R. S. Tipson, editors, J. Wileyand Sons, New York, 895-900 (1978) and references therein; R. Duschinskyin Nucleic Acid Chemistry, Part 1, L. B. Townsend and R. S. Tipson,editors, J. Wiley and Sons, Rev York, 43-46 (1978) and referencestherein). The fluorinating agent may be, for example,trimethylhypofluorite in fluorotrichloromethane.

With regard to process c), the compound of formula (IIC) isadvantageously treated with 1,2,4-triazole, advantageously together with4-chlorophenyl dichlorophosphate, to form the corresponding4-(1,2,4-triazolyl) compound which is then converted to the desired4-amino (cytidine) compound by reaction with for example methanol.

The starting materials of formula (IIB) and (IIC) may be prepared forexample by reaction of an appropriate (optionally protected) base with acompound of formula IIA in an analogous manner to that described inprocess a). 5-Fluorouracil and 5-fluorocytosine are commerciallyavailable from Aldrich Chemical Co., Milwaukee, Wis. 53233, USA.

Separation of the (±)-cis and (±)-trans isomers for example in aprotected form, may be accomplished by chromatography on silica gel withmixtures of organic solvents such as ethyl acetate/methanol,ethylacetate/hexane or dichloromethane/methanol. Any protecting groupmay then be removed using the appropriate reagent for each group.

The compound of formula (I) may be converted into a pharmaceuticallyacceptable esters and amides by reaction with an appropriate acylatingagent, for example, an acid halide or anhydride serving to acylate the5′-OH and 4-NH₂ groups. The acyl group may then be removed selectivelyfrom one or other of the 5′-OH and 4-NH₂ groups. For, example, treatmentof the diacylated compound under acidic conditions, eg. a Lewis acidsuch as zinc bromide in methanol, removes the 4N-acyl group to yield thecorresponding 5′-OH ester where treatment of the diacylated compoundunder alkaline conditions, eg. with sodium methoxide removes the 5′O—acyl group to yield the corresponding 4N-amide. The acyl groups can alsobe removed selectively by treatment with commercially available esteraseor lipase enzymes, eg. pig liver esterase or pancreatic lipase, or bytreatment in accordance with methods described in U.S. Pat. No.5,071,983. The compound of formula (I) may be converted into apharmaceutically acceptable salt thereof in a conventional manner, forexample, by treatment with an appropriate base. An ester or salt of acompound of formula (I) may be converted into the parent compound, forexample, by hydrolysis.

For a better understanding of the invention, the following Examples aregiven by way of illustration.

EXAMPLE 1cis-1-(2-(Hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluoro-cytosine

Method A: (±)-cis and (±)-trans2-benzoyloxymethyl-5-(N-4-acetyl-cyto-sin-1-yl)-1,3-oxathiolane areprepared and separated to the (±)-cis and (±) trans isomers as describedin European Patent Specification 0 382 526. (See U.S. Pat. No.5,047,407.) The (±)-cis isomer is fluorinated with trifluoromethylhypofluorite in fluorotrichloromethane (CCI₃F) and chloroform at −78°C., according to the method of Robins, et al. Nucleic Acid Chemistry,Part 2, 895-900, 1978. The N₄-acetyl and 2-benzoyl groups are removedwith dimethylamine in ethanol, and the product,(±)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine, isisolated.

Method B: (±)-cis and(±)-trans-2-benzoyloxymethyl-5-(uracil-1-yl)-1,3-oxathiolane areprepared as described in EP 0 382 526. After deprotection of the2-hydroxyl group with saturated methanolic ammonia, the isomers areseparated on silica gel using EtOAc/MeOH as eluant (EP 0 382 526). The(±)-cis isomer is reacted with acetic anhydride in pyridine at roomtemperature to give the 2-acetate. Solvent is removed in vacuo at <30°C. The 2-acetate is then dissolved in CHCl₃ and washed with aqueoussodium bicarbonate. The separated organic layer is dried, and CHCl₃ isevaporated in vacuo.(±)-cis-2-Acetylozymethyl-5-(uracil-1-yl)-1,3-oxathiolane isfluorina-ted as described above (Method A) by the method of Robins etal. Conversion of the 5-F-uracil base to the 5-F-cytosine base iscarried out by preparation of the 4-(1,2,4-triazol-1-yl) derivativeaccording to the methods of C. B. Reese, J. Chem. Soc., Perkins I, 1171,1984 and W. L. Sung, Nucleic Acids Res. 9:6139, 1981, using1,2,4-triazole and 2 equivalents of 4-chlorophenyldichlorophosphate indry pyridine at ambient temperature. This conversion is followed byreaction with methanol previously saturated with ammonia at 0° C., andthe 2-acetate is hydrolyzed to give(±)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine.

Pharmaceutical Formulations

In the following formulation Examples, the “Active Ingredient” iscis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine.

EXAMPLE 2 Table Formulations

The following formulations A, B and C are prepared by wet granulation ofthe ingredients with a solution of povidone, followed by addition ofmagnesium stearate and compression.

Formulation A mg/tablet mg/tablet (a) Active ingredient 250 250 (b)Lactose B.P. 210 26 (c) Povidone B.P. 15 9 (d) Sodium Starch Glycollate20 12 (e) Magnesium Stearate 5 3 500 300

Formulation B mg/tablet mg/tablet (a) Active ingredient 250 250 (b)Lactose 150 — (c) Avicel PH 101 60 26 (d) Povidone B.P. 15 9 (e) SodiumStarch Glycollate 20 12 (f) Magnesium Stearate 5 3 500 300

Formulation C mg/tablet Active ingredient 100 Lactose 200 Starch 50Povidone 5 Magnesium Stearate 4 359

The following formulations, D and E, are prepared by direct compressionof the admixed ingredients. The lactose in formulation E is of thedirect compression type (Dairy Crest-“Zeparoax”).

Formulation D mg/tablet Active ingredient 250 Pregelatinized Starch NF15150 400

Formulation E mg/tablet Active ingredient 250 Lactose 150 Avicel 100 500

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients(below) with a solution of povidone followed by the addition ofmagnesium stearate and compression.

mg/tablet (a) Active ingredient 500 (b) Hydroxypropylmethylcellulose 112(Methocel K4M Premium) (c) Lactose B.P. 53 (d) Povidone B.P. 28 (e)Magnesium Stearate 7 700

Drug release takes place over a period of about 6-8 hours and iscomplete after 12 hours.

EXAMPLE 3 Capsule Formulations Formulation A

A capsule formulation is prepared by admixing the ingredients ofFormulation D in Example 2 above and filling into a two-part hardgelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B mg/capsule (a) Active ingredient 250 (b) Lactose B.P. 143(c) Sodium Starch Glycollate 25 (d) Magnesium Stearate 2 420

Formulation C mg/capsule (a) Active ingredient 250 (b) Macrogol 4000B.P. 350 600

Formulation D mg/capsule Active ingredient 250 Lecithin 100 Arachis Oil100 450

Capsules of formulation D are prepared by dispersing the activeingredient in the lecithin and arachis oil and filling the dispersioninto soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared byextruding ingredients a, b and c using an extruder, followed byspheronization of the extrudate and drying. The dried pellets are thencoated with controlled-release membrane (d) and filled into a two-piece,hard gelatin capsule.

mg/capsule (a) Active ingredient 250 (b) Microcrystalline Cellulose 125(c) Lactose B.P. 125 (d) Ethyl Cellulose 13 513

EXAMPLE 4 Injectable Formulation

Formulation A Active ingredient 0.200 g Hydrochloric acid solution, 0.1M, or Sodium hydroxide solution, 0.1 M q.s. to pH 4.0 to 7.0 Sterilewater q.s. to 10 mL

The active ingredient is dissolved in most of the water (35° C.-40° C.)and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid orthe sodium hydroxide as appropriate. The batch is then made up to volumewith the water and filtered through a sterile micropore filter into asterile 10 μL amber glass vial (type 1) and sealed with sterile closuresand overseals.

Formulation B Active ingredient 0.125 Sterile, pyrogen-free, pH 7phosphate q.s. to 25 mL Buffer,

EXAMPLE 5

Intramuscular injection Active ingredient 0.20 g Benzyl Alcohol 0.10 gGlycofurol 75 1.45 g Benzyl Alcohol q.s. to 3.00 mL

The active ingredient is dissolved in the glycofurol. The benzyl alcoholis then added and dissolved, and water added to 3 mL. The mixture isthen filtered through a sterile micropore filter and sealed in sterile 3mL amber glass vials (type 1).

EXAMPLE 6

Syrup Active ingredient 0.25 g Sorbitol Solution 1.50 g Glycerol 2.00 gSodium Benzoate 0.005 g Flavor, Peach 17.42.316 g 0.0125 mL PurifiedWater q.s. to 5.00 mL

The active ingredient is dissolved in a mixture of the glycerol and mostof the purified water. An aqueous solution of the sodium benzoate isthen added to the solution, followed by addition of the sorbitolsolution and finally the flavor. The volume is made up with purifiedwater and mixed well.

EXAMPLE 7

Suppository mg/suppository Active ingredient 250 Hard Fat, B.P.(Witepsol H15 - Dynamit Nobel) 1770 2020

One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C. maximum. The active ingredient is sifted through a 200 μm sieve andadded to the molten base with mixing, using a Silverson fitted with acutting head, until smooth dispersion is achieved. Maintaining themixture at 45° C., the remaining Witepsol H15 is added to the suspensionand stirred to ensure a homogenous mix. The entire suspension is passedthrough a 250 μm stainless steel screen and, with continuous stirring,is allowed to cool to 40° C. At a temperature of 38° C. to 40° C., 2.02g of the mixture is filled into suitable, 2 mL plastic molds. Thesuppositories are allowed to cool to room temperature.

EXAMPLE 8

Pessaries mg/pessary Active ingredient 250 Anhydrate Dextrose 380 PotatoStarch 363 Magnesium Stearate 7 1000

The above ingredients are mixed directly and pessaries prepared bydirect compression of the resulting mixture.

EXAMPLE 9 Antiviral Activity Against Heratitis B Virus (HBV)

The compoundcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluoro-cytosine, wastested as described below.

The human HBV producer cell line of HepG₂, 2.2.15, described andcharacterized by Sells et al., PNAS 84:1005, 1987 and J. Virol. 62:2836,1988, has been shown to share many characteristics of the HBVchronically infected hepatocyte. It is infectious as demonstrated by theability to cause disease in chimpanzees. This cell line was utilized invitro to identify compounds with anti-HBV activity.

To test compounds for antiviral activity, monolayer cultures weretreated with compound, 50-2001M for ten days. Supernatant mediacontaining extracellular virion DNA (Dane particles) were harvested ondays three, six and ten, treated with proteinase K (1 mg/mL) and sodiumdodecyl sulfate (1%), and incubated at 50° C. for one hour. DNA wasextracted with equal volumes of phenol followed by chloroform and thenprecipitated by ammonium acetate and propanol. The DNA precipitate wasdissolved and collected on nitrocellulose using the procedure ofSchleicher and Schuell (S & S, 10 Optical Ave., Keene, N. H. 03431,Publication No. 700, 1987), and treated as described by Southern, J.Mol. Biol. 98:503, 1975. Cells were harvested, and the intracellular DNAwas obtained after cell lysis with guanidine isothiocyanate. Theintracellular DNA was handled in the same manner as the extracellularDNA. After precipitation by ammonium acetate and propanol, theintracellular DNA precipitate was dissolved, cut by restrictionendonuclease, Hind III, applied to agarose gel and then treated asdescribed by Southern to determine the quantity of replicativeintermediate forms. The antiviral effect of the compound was determinedby measuring at least a 100-fold reduction of the amount of Daneparticles extruded into the culture medium and a similar decrease in theintracellular replicative intermediates.

The results are given below:

Effect of cis-1-(2-(Hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosineon HBV Production in 2.2.15 Cell Cultures Intracellular HBV DNA* HBV DHAin Culture Medium Treatment (pg/μg cell DNA) (pg/mL)⁺+ CompoundReplicative Day Day Day Day (μM) Integrated Monomer- intermediate 0 3 610 A. untreated 1.1 2.0 81 58 67 93 77 cells 0.9 2.3 77 89 110 100 88100 1.9 0.8 2 64 11 3 0 1.5 1.9 1 34 19 2 0 B. untreated 1.5 1.9 110 6544 86 71 cells 1.0 2.3 67 90 120 80 82 100 1.6 0.8 1 90 16 0 0 1.0 0.7 174 10 0 0 *Analysis of intracellular HBV DNA (Dane particles) was 24hours following the 10th day of treatment. ⁺+A “zero” indicates anundetectable level of HBV DNA, sensitivity cutoff was 0.1 pg/mL

1-3. (canceled)
 4. A pharmaceutical composition that provides slow orcontrolled release comprising an effective amount ofcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine derivative offormula I

and a pharmaceutically acceptable carrier in a tablet or capsuleformulation.
 5. The composition of claim 4, wherein the formulationprovides slow release.
 6. The composition of claim 4, wherein theformulation provides controlled release.
 7. The composition of claim 4,in a tablet formulation.
 8. The composition of claim 7, wherein thetablet includes an enteric coating.
 9. The composition of claim 4, in acapsule formulation.
 10. The composition of claim 9, wherein the capsuleis a gelatin capsule.
 11. The composition of claim 10, wherein thederivative is coated with a controlled release membrane.
 12. Thecomposition of claim 11, wherein the controlled release membrane isethylcellulose
 13. The composition of claim 11, wherein the derivativecoated with the controlled release membrane is filled into the gelatincapsule.
 14. The composition of claim 4, wherein the formulationadditionally comprises hydroxypropyl methylcellulose.
 15. Thecomposition of claim 4, wherein the release occurs over 6 to 8 hours.16. The composition of claim 4, wherein the release is complete at 12hours.